Single base substitution SNP refers to a single base change in the base sequence in human DNA said to occur once in from 1000 bp to 2000 bp. It is estimated that adult humans, whether healthy or sick, have from 100,000 to several million SNPs.
Plural base substitution refers to a change in a number of bases in the base sequence of a gene.
Point mutation refers to a single-base change in the base sequence in an already known gene. It is by this that functional anomalies in translated protein are seen, and [this] sometimes becomes a cause of disease.
Gene translocation refers to the partial reversal of the order in a base sequence, which sometimes becomes a cause of disease.
Gene loss refers to a partial deficiency in a base sequence, which sometimes becomes a cause of disease.
Gene amplification refers to the multiplication of a portion of a base sequence, which sometimes becomes a cause of disease.
Triplet repeat refers to three base pairs repeating and growing, which sometimes becomes a cause of disease.
Means for detecting and analyzing differences in the base sequences in genetic DNA include DNA sequencing (base sequence determination method), the PCR-SSCP (polymerase chain reaction—single stranded polymorphism) method, the allele-specific hybridization method, the DNA chip method, and etc.
In DNA sequencing, there is the Maxam-Gilbert method and the Sanger (dideoxy) method, with the latter being principally used today. After amplifying the region of the human gene to be analyzed by the PCR (polymerase chain reaction) method, sequencing is performed using either the primer used in the PCR method or a primer set inside the amplified DNA, and the nucleotide sequence inside that region is determined.
With the PCR-SSCP (polymerase chain reaction—single stranded polymorphism) method, after using the PCR method to amplify the region of the human gene to be analyzed, this is made into single strand by thermal denaturation, and, by performing electrophoresis thereon in a non-denaturing polyacrylamide gel, a two-dimensional structure is formed (by intramolecular hydrogen bonds) in each strand of the two-strand DNA amplified by the PCR method. Because the two-dimensional structures will differ due to differences in the sequences, single base substitution SNPs and point mutations and the like are detected by differences in electrophoretic distance.
With the allele-specific hybridization method, oligonucleotide probes or PCR products of 20 bases or so are immobilized to a region of a membrane (nylon filter), and after amplifying the region to be analyzed by the PCR method, this sample DNA is labeled with the radioactive isotope 32P and hybridized. By adjusting the hybridization conditions such as temperature and the like at this time, SNPs and point mutations are detected by differences in the strength of the radioactive isotope.
With the DNA chip method, although in principle this is roughly the same as the allele-specific hybridization method, the oligonucleotide probes or PCR products for 20 bases or so are aligned in a stationary phase (on a substrate), and there the fluorescence labeled sample DNA is hybridized. By adjusting the hybridization conditions such as temperature and the like, the SNPs and the like in human genes are detected by differences in the intensity of fluorescence.
In the case of hybridization in the allele-specific hybridization method, because DNA is labeled with a radioactive isotope, the enormous cost involved in handling and controlling the radioactive isotopes becomes a problem. In the case of the DNA chip method, when labeling is done with a fluorescent radical, fluorescence is not incorporated into the DNA with adequate frequency due to the large molecular structure of the fluorescent radical, wherefore the fluorescent strength of the fluorescence labeled probe is not high, and other problems are encountered, namely fluorescence fading and the fluorescence exhibited by the glass or other platform (background fluorescence).
In order to resolve problems as these, as methods for detecting DNA hybrid formation and detecting two-strand DNA that are simple but exhibit outstanding sensitivity, methods have been disclosed wherewith the probe DNA is fixed to an electrode, that probe DNA is caused to react with sample DNA, two-strand DNA is detected in the presence of an intercalator, and the detection of the hybrid formation is performed electrochemically (cf. Japanese Patent Application Laid-Open No. H9-288080/1997 (published) and Dai57kai bunseki kagaku toronkai yokoshu [57th analytical chemistry debate manuscript collection], pp 137 and 138, 1996).
However, the number of gene single base substitution SNPs and gene mutations and the like are enormous. In the case of humans, for example, in order to produce a single base substitution SNP map with a 15 KB density (resolution), at least 2 million single base substitution SNPs must be identified. The number of gene mutations involved in known diseases is also extremely large. It is virtually impossible, realistically, to comprehensively analyze single base substitutions and point mutations with the conventional methods.
An object of the present invention is to resolve the problems with the prior art noted in the foregoing. To that end, the present invention provides a gene identification apparatus capable of detecting and analyzing large volumes of genes, for a plurality of sample DNA, that is, a gene identification apparatus capable of high throughput (fast and high volume) processing, and also capable of performing high-sensitivity detection and analysis. In fine, the present invention seeks to realize a high-volume, high-sensitivity gene detection and analysis apparatus that is based on the principle of electrochemically performing two-strand DNA detection and hybrid formation detection described in Japanese Patent Application Laid-Open No. H9-288080/1997 (published).
The present invention also seeks to realize a gene detection method, detection apparatus, and detecting chip that, in actual operations involving detection and the like, feature ease of handling and good workability.